Influenza virus-like particles (VLPs) have demonstrated in preclinical studies that they are highly immunogenic and efficacious in protecting against influenza infections. This emerging vaccine technology not only creates new vaccine candidates with alternative routes of administration (intranasal or intramuscular) but also implements a fast, flexible, and dependable cell-based manufacturing system that overcomes many of the burdens associated with the current egg production system. VLPs are structures that morphologically and biochemically mimic a wild type influenza virus, however they lack viral genetic material and are unable to replicate and cause infection;therefore chemical inactivation is not required. This better maintains the native conformation of the antigenic epitopes in the VLP vaccine. The safety of the technology allows for the development of vaccines directed against highly pathogenic influenza virus strains without the risk of disseminating infectious material. The Phase I SBIR award allowed us to create and test in preclinical studies several VLP vaccine candidates designed to protect against highly pathogenic pandemic strains of the influenza virus such as H1N1-1918;H5N1 and H7N7 avian influenza viruses. These studies have shown that VLP vaccines are immunogenic and efficacious, further proving the value of the VLP technology for influenza vaccine development. In the current application, we propose to further advance the development of these vaccine products with additional preclinical studies. We will develop a purification system for large scale manufacturing of VLP vaccines, prior to cGMP manufacturing. We will perform one comparative pilot study between a VLP vaccine produced in the insect cell/baculovirus system and the same VLP vaccine produced in stably transfected recombinant mammalian cells. Further, we aim to perform small scale cGMP vaccine production and toxicology studies for a Pre-Innovation Drug Application (IND) with the FDA. The specific aims of the Phase II SBIR are: A) To produce, characterize, and test in preclinical studies in mice the influenza-virus-like particle (VLP) vaccines designed to protect against 1) H5N1 avian influenza virus, 2) human H1N1-1918 influenza virus and 3) H7N7 avian influenza, and to perform cross protection and enhancement of immunogenicity (including in the elderly) studies as well as to develop a large scale purification method and to compare the immunogenicity of the H5N1 VLP vaccine produced in the insect cell/ baculovirus system and the same vaccine produced in a recently developed stably transformed mammalian which could be developed as a second generation VLP manufacturing system. B) To evaluate vaccine efficacy, dosage and adjuvant formulations for enhancement of vaccine potency and antigen sparing in preclinical studies in ferrets. C) Perform small scale cGMP vaccine production and toxicology studies for a Pre-Innovation Drug Application (IND) with the FDA. Completion of this SBIR Phase II will bring the VLP vaccine technology to the point of a Phase I clinical trial in humans and will advance this new vaccine product and manufacturing system towards commercialization. Although pandemic influenza VLP vaccines are being developed during this project, the outcome will validate the robustness of the technology for development of seasonal influenza vaccine which currently is burdened with prolonged production times, dependence on chicken eggs, insufficient yield with certain strains, inflexibility for accommodating newly emerging variants, and unsuitability when working with highly virulent virus, all issues where the VLP technology is superior. This SBIR Phase II will accelerate the creation of vital prophylactic vaccines (e.g. H5, H7 avian flu and 1918) and will also validate a faster, more flexible, cell-based technology for epidemic influenza vaccine production. PUBLIC HEALTH RELEVANCE: Influenza is a recurrent respiratory infection afflicting humans all over the world. The virus changes constantly generating new epidemic and pandemic viruses, for which vaccines are needed. Although prophylactic vaccines for epidemic strains are available, periodic updates are required to maintain protective efficacy. Vaccines for pandemic viruses are lacking and their development is critical. Here we proposed to further develop the influenza virus-like particle (VLP) technology for the generation of pandemic and epidemic influenza vaccines. This technology is fast, flexible, and reliable for creating highly effective vaccines which are manufactured in a cell-based system. The VLP vaccines system overcomes many of the problems of the egg production system. Successful development of these vaccines will benefit society globally.